Novel human carboxypeptidases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/156,685 which was filed on Sep. 29, 1999 and isherein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding proteinsthat share sequence similarity with animal proteases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or over express the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed sequences that can be used for diagnosis, drug screening,clinical trial monitoring and the treatment of physiological disorders.

2. BACKGROUND OF THE INVENTION

[0003] Proteases are enzymes that cleave polypeptide sequences.Carboxypeptidases are proteases that hydrolyze the peptide bonds at thecarboxy-terminal end of a chain of amino acids and have been identifiedin a wide variety of cell types and animals. Peptidases have beenimplicated in a wide variety of biological processes including, but notlimited to, digestion, coagulation, diabetes, prostate cancer,gynecological disorders, neurological disorders, and obesity.Accordingly, peptidases represent significant targets for regulatorycontrol of a variety of physiological processes and pathways.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel human proteins,and the corresponding amino acid sequences of these proteins. The novelhuman proteins (NHPs) described for the first time herein sharestructural similarity with animal proteases, and especiallycarboxypeptidases. As such, the described NHPs represent a new family ofprotease-related proteins with a range of homologues and orthologs thattranscend phyla and a broad range of species.

[0005] The novel human nucleic acid sequences described herein, encodeproteins/open reading frames (ORFs) of 47, 88, 247, 92, 437, and 350amino acids in length (see SEQ ID NOS: 2, 4, 6, 8, 10, and 12respectively).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHPs, including small molecules, large molecules, mutant NHPs,or portions thereof that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and gene or regulatory sequence replacement constructs) or toenhance the expression of the described NHP sequences (e.g., expressionconstructs that place the described sequence under the control of astrong promoter system), and transgenic animals that express a NHPtransgene, or “knock-outs” (which can be conditional) that do notexpress a functional NHP.

[0007] Further, the present invention also relates to processes ofidentifying compounds that modulate, i.e., act as agonists orantagonists, of NHP expression and/or NHP product activity that utilizepurified preparations of the described NHPs and/or NHP product, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0008] The Sequence Listing provides the sequences of 6 protease-relatedORFs that encode the described NHP amino acid sequences.

5. DETAILED DESCRIPTION OF THE INVENTION

[0009] The NHPs, described for the first time herein, are novel proteinsthat are expressed in, inter alia, human cell lines, and human brain,pituitary, spinal cord, thymus, spleen, lymph node, bone marrow,trachea, lung, kidney, prostate, testis, thyroid, adrenal gland,stomach, small intestine colon, skeletal muscle, uterus, mammary gland,bladder, and cervix cells. The described sequences were compiled fromgene trapped cDNAs and a clone isolated from a human prostate cDNAlibrary (Edge Biosystems, Gaithersburg, Md.). The present inventionencompasses the nucleotides presented in the Sequence Listing, hostcells expressing such nucleotides, the expression products of suchnucleotides, and: (a) nucleotides that encode mammalian homologs of thedescribed genes, including the specifically described NHPs, and the NHPproducts; (b) nucleotides that encode one or more portions of the NHPsthat correspond to functional domains, and the polypeptide productsspecified by such nucleotide sequences, including but not limited to thenovel regions of any active domain(s); (c) isolated nucleotides thatencode mutant versions, engineered or naturally occurring, of thedescribed NHPs in which all or a part of at least one domain is deletedor altered, and the polypeptide products specified by such nucleotidesequences, including but not limited to soluble proteins and peptides inwhich all or a portion of the signal sequence in deleted; (d)nucleotides that encode chimeric fusion proteins containing all or aportion of a coding region of an NHP, or one of its domains (e.g., areceptor/ligand binding domain, accessory protein/self-associationdomain, etc.) fused to another peptide or polypeptide; or (e)therapeutic or diagnostic derivatives of the described polynucleotidessuch as oligonucleotides, antisense polynucleotides, ribozymes, dsRNA,or gene therapy constructs comprising a sequence first disclosed in theSequence Listing.

[0010] As discussed above, the present invention includes: (a) the humanDNA sequences presented in the Sequence Listing (and vectors comprisingthe same) and additionally contemplates any nucleotide sequence encodinga contiguous NHP open reading frame (ORF) that hybridizes to acomplement of a DNA sequence presented in the Sequence Listing underhighly stringent conditions, e.g., hybridization to filter-bound DNA in0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1× SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., NY, at p. 2.10.3) andencodes a functionally equivalent gene product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of the DNA sequence that encode and express an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2× SSC/0.1% SDS at 42° C. (Ausubel etal., 1989, supra), yet still encode a functionally equivalent NHPproduct. Functional equivalents of a NHP include naturally occurringNHPs present in other species and mutant NHPs whether naturallyoccurring or engineered (by site directed mutagenesis, gene shuffling,directed evolution as described in, for example, U.S. Pat. No.5,837,458). The invention also includes degenerate nucleic acid variantsof the disclosed NHP polynucleotide sequences.

[0011] Additionally contemplated are polynucleotides encoding NHP ORFs,or their functional equivalents, encoded by polynucleotide sequencesthat are about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NO:1 (as measured by BLAST sequence comparisonanalysis using, for example, the GCG sequence analysis package usingdefault parameters).

[0012] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP gene nucleotide sequences. Such hybridizationconditions may be highly stringent or less highly stringent, asdescribed above. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80, or about 34 toabout 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0013] Alternatively, such NHP oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a micro array or high-throughput“chip” format). Additionally, a series of the described NHPoligonucleotide sequences, or the complements thereof, can be used torepresent all or a portion of the described NHP sequences. Theoligonucleotides, typically between about 16 to about 40 (or any wholenumber within the stated range) nucleotides in length may partiallyoverlap each other and/or the NHP sequence may be represented usingoligonucleotides that do not overlap. Accordingly, the described NHPpolynucleotide sequences shall typically comprise at least about two orthree distinct oligonucleotide sequences of at least about 18, andpreferably about 25, nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences maybegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

[0014] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6× SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP sequence antisense molecules, useful, forexample, in NHP sequence regulation (for and/or as antisense primers inamplification reactions of NHP nucleic acid sequences). With respect toNHP sequence regulation, such techniques can be used to regulatebiological functions. Further, such sequences may be used as part ofribozyme and/or triple helix sequences that are also useful for NHPsequence regulation.

[0015] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0016] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0017] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0018] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

[0019] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0020] Low stringency conditions are well known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Spring Harbor Press, NY; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, NY.

[0021] Alternatively, suitably labeled NHP nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0022] Further, a NHP sequence homolog can be isolated from nucleic acidfrom an organism of interest by performing PCR using two degenerate or“wobble” oligonucleotide primer pools designed on the basis of aminoacid sequences within the NHP products disclosed herein. The templatefor the reaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue known or suspected to express an allele of a NHPsequence.

[0023] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0024] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express a NHP sequence, such as, for example,testis tissue). A reverse transcription (RT) reaction can be performedon the RNA using an oligonucleotide primer specific for the most 5′ endof the amplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” using a standard terminaltransferase reaction, the hybrid may be digested with RNase H, andsecond strand synthesis may then be primed with a complementary primer.Thus, cDNA sequences upstream of the amplified fragment can be isolated.For a review of cloning strategies that can be used, see e.g., Sambrooket al., 1989, supra.

[0025] A cDNA encoding a mutant NHP sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand can besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known or suspected to be expressed in an individualputatively carrying a mutant NHP allele, and by extending the new strandwith reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal gene. Using these two primers, the product is thenamplified via PCR, optionally cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well known to thoseof skill in the art. By comparing the DNA sequence of the mutant NHPallele to that of a corresponding normal NHP allele, the mutation(s)responsible for the loss or alteration of function of the mutant NHPsequence product can be ascertained.

[0026] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of or known to carry a mutant NHPallele (e.g., a person manifesting a NHP-associated phenotype such as,for example, obesity, high blood pressure, etc.), or a cDNA library canbe constructed using RNA from a tissue known, or suspected, to express amutant NHP allele. A normal NHP sequence, or any suitable fragmentthereof, can then be labeled and used as a probe to identify thecorresponding mutant NHP allele in such libraries. Clones containingmutant NHP coding sequences can then be purified and subjected tosequence analysis according to methods well known to those skilled inthe art.

[0027] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHP allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHP product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.)

[0028] Additionally, screening can be accomplished using labeled NHPfusion proteins, such as, for example, alkaline phosphatase -NHP orNHP-alkaline phosphatase fusion proteins. In cases where a NHP mutationresults in an expressed gene product with altered function (e.g., as aresult of a missense or a frameshift mutation), polyclonal antibodies toa NHP are likely to cross-react with a corresponding mutant NHP sequenceproduct. Library clones detected via their reaction with such labeledantibodies can be purified and subjected to sequence analysis accordingto methods well known in the art.

[0029] The invention also encompasses (a) DNA vectors that contain anyof the foregoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the humancytomegalovirus (hCMV) immediate early gene, regulatable, viral elements(particularly retroviral LTR promoters), the early or late promoters ofSV40 adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast a-mating factors.

[0030] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of the NHP, as well as compounds or nucleotide constructs thatinhibit expression of a NHP sequence (transcription factor inhibitors,antisense and ribozyme molecules, or gene or regulatory sequencereplacement constructs), or promote the expression of a NHP (e.g.,expression constructs in which NHP coding sequences are operativelyassociated with expression control elements such as promoters,promoter/enhancers, etc.).

[0031] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHPs or inappropriately expressed NHPs for thediagnosis of disease. The NHP proteins or peptides, NHP fusion proteins,NHP nucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of NHP inthe body. The use of engineered host cells and/or animals may offer anadvantage in that such systems allow not only for the identification ofcompounds that bind to an endogenous receptor/ligand of a NHP, but canalso identify compounds that trigger NHP-mediated activity.

[0032] Finally, the NHP products can be used as therapeutics. Forexample, soluble versions or derivatives of a NHP, or peptides/domainscorresponding a NHP, NHP fusion protein products (especially NHP-Igfusion proteins, i.e., fusions of a NHP, or a domain of a NHP, to anIgFc), NHP antibodies and anti-idiotypic antibodies (including Fabfragments), antagonists or agonists (including compounds that modulateor act on downstream targets in a NHP-mediated pathway) can be used todirectly treat diseases or disorders. For instance, the administrationof an effective amount of soluble NHP, or a NHP-IgFc fusion protein oran anti-idiotypic antibody (or its Fab) that mimics the NHP couldactivate or effectively antagonize NHP function. Nucleotide constructsencoding such NHP products can be used to genetically engineer hostcells to express such products in vivo; these genetically engineeredcells function as “bioreactors” in the body delivering a continuoussupply of a NHP, a NHP peptide, or a NHP fusion protein to the body.Nucleotide constructs encoding functional NHPs, mutant NHPs, as well asantisense and ribozyme molecules can also be used in “gene therapy”approaches for the modulation of NHP expression. Thus, the inventionalso encompasses pharmaceutical formulations and methods for treatingbiological disorders.

[0033] Various aspects of the invention are described in greater detailin the subsections below.

5.1 The NHP Sequences

[0034] The cDNA sequences and the corresponding deduced amino acidsequences of the described NHPs are presented in the Sequence Listing.The NHP sequences were obtained from a human prostate cDNA library usingprobes and/or primers generated from human gene trapped sequence tags.Expression analysis has provided evidence that the described NHPs can beexpressed, for example, in a variety of human cell types and that thedescribed NHPs share significant similarity to a variety of proteases,and especially carboxypeptidase B or carboxypetidase A, from, interalia, humans, mice, and rats. SEQ ID NO: 13 describes a full length NHPORF with flanking 5′ and 3′ sequences.

5.2 NHPS and NHP Polypeptides

[0035] NHPs, polypeptides, peptide fragments, mutated, truncated, ordeleted forms of the NHPs, and/or NHP fusion proteins can be preparedfor a variety of uses. These uses include, but are not limited to, thegeneration of antibodies, as reagents in diagnostic assays, for theidentification of other cellular gene products related to a NHP, asreagents in assays for screening for compounds that can be aspharmaceutical reagents useful in the therapeutic treatment of mental,biological, or medical disorders and disease. Several uses andapplications for plasma carboxypeptidases similar to those describedherein are described in U.S. Pat. No. 5,593,674, the disclosure of whichis herein incorporated by reference in its entirety.

[0036] The Sequence Listing discloses the amino acid sequences encodedby the described NHP polynucleotides. The NHPs have initiatormethionines in DNA sequence contexts consistent with a translationinitiation site and a hydrophobic signal-like sequence is present nearthe N-terminal region of the protein. The sequence data presented hereinindicate that alternatively spliced forms of the NHPs exist (which mayor may not be tissue specific).

[0037] The NHP amino acid sequences of the invention include thenucleotide and amino acid sequences presented in the Sequence Listing aswell as analogues and derivatives thereof. Further, corresponding NHPhomologues from other species are encompassed by the invention. In fact,any NHP protein encoded by the NHP nucleotide sequences described above,are within the scope of the invention, as are any novel polynucleotidesequences encoding all or any novel portion of an amino acid sequencepresented in the Sequence Listing. The degenerate nature of the geneticcode is well known, and, accordingly, each amino acid presented in theSequence Listing, is generically representative of the well knownnucleic acid “triplet” codon, or in many cases codons, that can encodethe amino acid. As such, as contemplated herein, the amino acidsequences presented in the Sequence Listing, when taken together withthe genetic code (see, for example, Table 4-1 at page 109 of “MolecularCell Biology”, 1986, J. Darnell et al. eds., Scientific American Books,New York, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0038] The invention also encompasses proteins that are functionallyequivalent to the NHPs encoded by the presently described nucleotidesequences as judged by any of a number of criteria, including, but notlimited to, the ability to bind and cleave a substrate of a NHP, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHPproteins include, but are not limited to, additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but which result in a silentchange, thus producing a functionally equivalent gene product. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0039] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. The presentlydescribed NHPs are similar to plasma carboxypeptidase B and are likelysoluble proteins. Where the NHP peptide or polypeptide to be expressedis a soluble NHP protein, or a NHP peptide derived from a substantiallynonhydrophobic domain of a NHP, or a truncated or deleted NHP thepeptide or polypeptide can be recovered from the culture, i.e., from thehost cell in cases where the NHP peptide or polypeptide is not secreted,or from the culture media in cases where the NHP peptide or polypeptideis secreted by the cells. However, such expression systems alsoencompass engineered host cells that express a NHP, or functionalequivalent, in situ, i.e., anchored in the cell membrane. Purificationor enrichment of a NHP from such expression systems can be accomplishedusing appropriate detergents and lipid micelles and methods well knownto those skilled in the art. However, such engineered host cellsthemselves may be used in situations where it is important not only toretain the structural and functional characteristics of the NHP, but toassess biological activity, e.g., in drug screening assays.

[0040] The expression systems that can be used for purposes of theinvention include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing NHPnucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformedwith recombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP sequences; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing NHP nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0041] In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. PGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target sequence productcan be released from the GST moiety.

[0042] In an insect system, Autographa californica nuclear polyhidrosisvirus (AcNPV) is used as a vector to express foreign sequences. Thevirus grows in Spodoptera frugiperda cells. A NHP coding sequence may becloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofNHP coding sequence will result in inactivation of the polyhedrin geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted sequence is expressed (e.g., see Smith et al.,1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0043] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHP nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHP product in infected hosts(e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHP nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHP sequence or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHP coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, must be provided. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (See Bittner et al.,1987, Methods in Enzymol. 153:516-544).

[0044] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes thesequence product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include, but are not limited to, CHO, VERO, BHK,HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, human cell lines.

[0045] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the NHP sequences described above may be engineered. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the NHP product. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds that affectthe endogenous activity of the NHP product.

[0046] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

[0047] Alternatively, any fusion protein may be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the sequence of interest is subcloned into avaccinia recombination plasmid such that the sequence's open readingframe is translationally fused to an amino-terminal tag consisting ofsix histidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺.nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

5.3 Antibodies to NHP Products

[0048] Antibodies that specifically recognize one or more epitopes of aNHP, or epitopes of conserved variants of a NHP, or peptide fragments ofa NHP are also encompassed by the invention. Such antibodies include butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above. Antibodies, and applications are usesthereof, similar to those contemplated herein are described in U.S. Pat.No. 5,474,901 the disclosure of which is herein incorporated byreference in its entirety.

[0049] The antibodies of the invention can be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes, asdescribed, below, in Section 5.5, for the evaluation of the effect oftest compounds on expression and/or activity of a NHP coding sequenceproduct. Additionally, such antibodies can be used in conjunction genetherapy to, for example, evaluate the normal and/or engineeredNHP-expressing cells prior to their introduction into the patient. Suchantibodies may additionally be used as a method for the inhibition ofabnormal NHP activity. Thus, such antibodies may, therefore, be utilizedas part of treatment methods.

[0050] For the production of antibodies, various host animals may beimmunized by injection with the NHP, an NHP peptide (e.g., onecorresponding the a functional domain of an NHP), truncated NHPpolypeptides (NHP in which one or more domains have been deleted),functional equivalents of the NHP or mutated variant of the NHP. Suchhost animals may include but are not limited to pigs, rabbits, mice,goats, and rats, to name but a few. Various adjuvants may be used toincrease the immunological response, depending on the host species,including but not limited to Freund's adjuvant (complete andincomplete), mineral salts such as aluminum hydroxide or aluminumphosphate, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, and potentially usefulhuman adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Alternatively, the immune response could beenhanced by combination and or coupling with molecules such as keyholelimpet hemocyanin, tetanus toxoid, diptheria toxoid, ovalbumin, choleratoxin or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0051] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

[0052] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures which are herein incorporatedby reference in their entirety.

[0053] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce single chain antibodies against NHP sequence products. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0054] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0055] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona, 1993,FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to a NHP domain andcompetitively inhibit the binding of NHP to its cognate receptor/ligandcan be used to generate anti-idiotypes that “mimic” the NHP and,therefore, bind and activate or neutralize a receptor, cofactor, ligand,or binding partner. Such anti-idiotypic antibodies or Fab fragments ofsuch anti-idiotypes can be used in therapeutic regimens involving a NHPmediated pathway.

[0056] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited puiblications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 13 1 141 DNA homo sapiens 1 atgaagtgtc tcgggaagcg caggggccaggcagctgctt tcctgcctct ttgctggctc 60 tttttgaaga ttctgcaacc ggggcacagccacctttata acaaccgcta tgctggtcca 120 cagggaaaga aacttttgaa a 141 2 47PRT homo sapiens 2 Met Lys Cys Leu Gly Lys Arg Arg Gly Gln Ala Ala AlaPhe Leu Pro 1 5 10 15 Leu Cys Trp Leu Phe Leu Lys Ile Leu Gln Pro GlyHis Ser His Leu 20 25 30 Tyr Asn Asn Arg Tyr Ala Gly Pro Gln Gly Lys LysLeu Leu Lys 35 40 45 3 264 DNA homo sapiens 3 atgaagtgtc tcgggaagcgcaggggccag gcagctgctt tcctgcctct ttgctggctc 60 tttttgaaga ttctgcaaccggggcacagc cacctttata acaaccgcta tgctggtgat 120 aaagtgataa gatttattcccaaaacagaa gaggaagcat atgcactgaa gaaaatatcc 180 tatcaactta aggttggttcctgcactaca ggtggacctg tggcagccca gcagtatctc 240 ctatgtatca gagggaacagttac 264 4 88 PRT homo sapiens 4 Met Lys Cys Leu Gly Lys Arg Arg Gly GlnAla Ala Ala Phe Leu Pro 1 5 10 15 Leu Cys Trp Leu Phe Leu Lys Ile LeuGln Pro Gly His Ser His Leu 20 25 30 Tyr Asn Asn Arg Tyr Ala Gly Asp LysVal Ile Arg Phe Ile Pro Lys 35 40 45 Thr Glu Glu Glu Ala Tyr Ala Leu LysLys Ile Ser Tyr Gln Leu Lys 50 55 60 Val Gly Ser Cys Thr Thr Gly Gly ProVal Ala Ala Gln Gln Tyr Leu 65 70 75 80 Leu Cys Ile Arg Gly Asn Ser Tyr85 5 741 DNA homo sapiens 5 atgaagtgtc tcgggaagcg caggggccag gcagctgctttcctgcctct ttgctggctc 60 tttttgaaga ttctgcaacc ggggcacagc cacctttataacaaccgcta tgctggtgat 120 aaagtgataa gatttattcc caaaacagaa gaggaagcatatgcactgaa gaaaatatcc 180 tatcaactta aggtggacct gtggcagccc agcagtatctcctatgtatc agagggaaca 240 gttactgatg tccatatccc ccaaaatggt tcccgagccctgttagcctt cttacaggaa 300 gccaacatcc agtacaaggt cctcatagaa gatcttcagaaaacactgga gaagggaagc 360 agcttgcaca cccagagaaa ccgaagatcc ctctctggatataattatga agtttatcac 420 tccttagaag aaattcaaaa ttggatgcat catctgaataaaactcactc aggcctcatt 480 cacatgttct ctattggaag atcatatgag ggaagatctctttttatttt aaagctgggc 540 agacgatcac gactcaaaag agctgtttgg atagactgtggtattcatgc aagagaatgg 600 attggtcctg ccttttgtca gtggtttgta aaagaagtcctagaaaacac agctcacaaa 660 tgtcaagaat gtactaaatt tacaaaatat ctctgccactaccaaaacca caaaagtatg 720 cttaatcttg taagtattga g 741 6 247 PRT homosapiens 6 Met Lys Cys Leu Gly Lys Arg Arg Gly Gln Ala Ala Ala Phe LeuPro 1 5 10 15 Leu Cys Trp Leu Phe Leu Lys Ile Leu Gln Pro Gly His SerHis Leu 20 25 30 Tyr Asn Asn Arg Tyr Ala Gly Asp Lys Val Ile Arg Phe IlePro Lys 35 40 45 Thr Glu Glu Glu Ala Tyr Ala Leu Lys Lys Ile Ser Tyr GlnLeu Lys 50 55 60 Val Asp Leu Trp Gln Pro Ser Ser Ile Ser Tyr Val Ser GluGly Thr 65 70 75 80 Val Thr Asp Val His Ile Pro Gln Asn Gly Ser Arg AlaLeu Leu Ala 85 90 95 Phe Leu Gln Glu Ala Asn Ile Gln Tyr Lys Val Leu IleGlu Asp Leu 100 105 110 Gln Lys Thr Leu Glu Lys Gly Ser Ser Leu His ThrGln Arg Asn Arg 115 120 125 Arg Ser Leu Ser Gly Tyr Asn Tyr Glu Val TyrHis Ser Leu Glu Glu 130 135 140 Ile Gln Asn Trp Met His His Leu Asn LysThr His Ser Gly Leu Ile 145 150 155 160 His Met Phe Ser Ile Gly Arg SerTyr Glu Gly Arg Ser Leu Phe Ile 165 170 175 Leu Lys Leu Gly Arg Arg SerArg Leu Lys Arg Ala Val Trp Ile Asp 180 185 190 Cys Gly Ile His Ala ArgGlu Trp Ile Gly Pro Ala Phe Cys Gln Trp 195 200 205 Phe Val Lys Glu ValLeu Glu Asn Thr Ala His Lys Cys Gln Glu Cys 210 215 220 Thr Lys Phe ThrLys Tyr Leu Cys His Tyr Gln Asn His Lys Ser Met 225 230 235 240 Leu AsnLeu Val Ser Ile Glu 245 7 276 DNA homo sapiens 7 atgaagtgtc tcgggaagcgcaggggccag gcagctgctt tcctgcctct ttgctggctc 60 tttttgaaga ttctgcaaccggggcacagc cacctttata acaaccgcta tgctggtgat 120 aaagtgataa gatttattcccaaaacagaa gaggaagcat atgcactgaa gaaaatatcc 180 tatcaactta agggtcctcatagaagatct tcagaaaaca ctggagaagg gaagcagctt 240 gcacacccag agaaaccgaagatccctctc tggata 276 8 92 PRT homo sapiens 8 Met Lys Cys Leu Gly LysArg Arg Gly Gln Ala Ala Ala Phe Leu Pro 1 5 10 15 Leu Cys Trp Leu PheLeu Lys Ile Leu Gln Pro Gly His Ser His Leu 20 25 30 Tyr Asn Asn Arg TyrAla Gly Asp Lys Val Ile Arg Phe Ile Pro Lys 35 40 45 Thr Glu Glu Glu AlaTyr Ala Leu Lys Lys Ile Ser Tyr Gln Leu Lys 50 55 60 Gly Pro His Arg ArgSer Ser Glu Asn Thr Gly Glu Gly Lys Gln Leu 65 70 75 80 Ala His Pro GluLys Pro Lys Ile Pro Leu Trp Ile 85 90 9 1311 DNA homo sapiens 9atgaagtgtc tcgggaagcg caggggccag gcagctgctt tcctgcctct ttgctggctc 60tttttgaaga ttctgcaacc ggggcacagc cacctttata acaaccgcta tgctggtgat 120aaagtgataa gatttattcc caaaacagaa gaggaagcat atgcactgaa gaaaatatcc 180tatcaactta aggtggacct gtggcagccc agcagtatct cctatgtatc agagggaaca 240gttactgatg tccatatccc ccaaaatggt tcccgagccc tgttagcctt cttacaggaa 300gccaacatcc agtacaaggt cctcatagaa gatcttcaga aaacactgga gaagggaagc 360agcttgcaca cccagagaaa ccgaagatcc ctctctggat ataattatga agtttatcac 420tccttagaag aaattcaaaa ttggatgcat catctgaata aaactcactc aggcctcatt 480cacatgttct ctattggaag atcatatgag ggaagatctc tttttatttt aaagctgggc 540agacgatcac gactcaaaag agctgtttgg atagactgtg gtattcatgc aagagaatgg 600attggtcctg ccttttgtca gtggtttgta aaagaagctc ttctaacata taagagtgac 660ccagccatga gaaaaatgtt gaatcatcta tatttctata tcatgcctgt gtttaacgtc 720gatggatacc attttagttg gaccaatgat cgattttgga gaaaaacaag gtcaaggaac 780tcaaggtttc gctgccgtgg agtggatgcc aatagaaact ggaaagtgaa gtggtgtgat 840gaaggagctt ctatgcaccc ttgtgatgac acatactgtg gcccttttcc agaatctgag 900ccggaagtga aggctgtagc taacttcctt cgaaaacaca gaaagcacat tagggcttat 960ctctcctttc atgcatatgc tcagatgtta ctgtatccct attcttacaa atatgcaaca 1020attcccaatt ttagatgtgt ggaatctgca gcttataaag ctgtgaatgc acttcagtca 1080gtatacgggg tacgatacag atatggacca gcctccacaa cgttgtatgt gagctctggt 1140agctcaatgg attgggccta caaaaatgga ataccttatg catttgcttt cgaactacgt 1200gacactggat attttggatt tttactccca gagatgctca tcaaacccac ctgtacagaa 1260actatgctgg ctgtgaaaaa tatcacaatg cacctgctaa agaaatgtcc c 1311 10 437 PRThomo sapiens 10 Met Lys Cys Leu Gly Lys Arg Arg Gly Gln Ala Ala Ala PheLeu Pro 1 5 10 15 Leu Cys Trp Leu Phe Leu Lys Ile Leu Gln Pro Gly HisSer His Leu 20 25 30 Tyr Asn Asn Arg Tyr Ala Gly Asp Lys Val Ile Arg PheIle Pro Lys 35 40 45 Thr Glu Glu Glu Ala Tyr Ala Leu Lys Lys Ile Ser TyrGln Leu Lys 50 55 60 Val Asp Leu Trp Gln Pro Ser Ser Ile Ser Tyr Val SerGlu Gly Thr 65 70 75 80 Val Thr Asp Val His Ile Pro Gln Asn Gly Ser ArgAla Leu Leu Ala 85 90 95 Phe Leu Gln Glu Ala Asn Ile Gln Tyr Lys Val LeuIle Glu Asp Leu 100 105 110 Gln Lys Thr Leu Glu Lys Gly Ser Ser Leu HisThr Gln Arg Asn Arg 115 120 125 Arg Ser Leu Ser Gly Tyr Asn Tyr Glu ValTyr His Ser Leu Glu Glu 130 135 140 Ile Gln Asn Trp Met His His Leu AsnLys Thr His Ser Gly Leu Ile 145 150 155 160 His Met Phe Ser Ile Gly ArgSer Tyr Glu Gly Arg Ser Leu Phe Ile 165 170 175 Leu Lys Leu Gly Arg ArgSer Arg Leu Lys Arg Ala Val Trp Ile Asp 180 185 190 Cys Gly Ile His AlaArg Glu Trp Ile Gly Pro Ala Phe Cys Gln Trp 195 200 205 Phe Val Lys GluAla Leu Leu Thr Tyr Lys Ser Asp Pro Ala Met Arg 210 215 220 Lys Met LeuAsn His Leu Tyr Phe Tyr Ile Met Pro Val Phe Asn Val 225 230 235 240 AspGly Tyr His Phe Ser Trp Thr Asn Asp Arg Phe Trp Arg Lys Thr 245 250 255Arg Ser Arg Asn Ser Arg Phe Arg Cys Arg Gly Val Asp Ala Asn Arg 260 265270 Asn Trp Lys Val Lys Trp Cys Asp Glu Gly Ala Ser Met His Pro Cys 275280 285 Asp Asp Thr Tyr Cys Gly Pro Phe Pro Glu Ser Glu Pro Glu Val Lys290 295 300 Ala Val Ala Asn Phe Leu Arg Lys His Arg Lys His Ile Arg AlaTyr 305 310 315 320 Leu Ser Phe His Ala Tyr Ala Gln Met Leu Leu Tyr ProTyr Ser Tyr 325 330 335 Lys Tyr Ala Thr Ile Pro Asn Phe Arg Cys Val GluSer Ala Ala Tyr 340 345 350 Lys Ala Val Asn Ala Leu Gln Ser Val Tyr GlyVal Arg Tyr Arg Tyr 355 360 365 Gly Pro Ala Ser Thr Thr Leu Tyr Val SerSer Gly Ser Ser Met Asp 370 375 380 Trp Ala Tyr Lys Asn Gly Ile Pro TyrAla Phe Ala Phe Glu Leu Arg 385 390 395 400 Asp Thr Gly Tyr Phe Gly PheLeu Leu Pro Glu Met Leu Ile Lys Pro 405 410 415 Thr Cys Thr Glu Thr MetLeu Ala Val Lys Asn Ile Thr Met His Leu 420 425 430 Leu Lys Lys Cys Pro435 11 1050 DNA homo sapiens 11 atgaagtgtc tcgggaagcg caggggccaggcagctgctt tcctgcctct ttgctggctc 60 tttttgaaga ttctgcaacc ggggcacagccacctttata acaaccgcta tgctggtgat 120 aaagtgataa gatttattcc caaaacagaagaggaagcat atgcactgaa gaaaatatcc 180 tatcaactta aggtggacct gtggcagcccagcagtatct cctatgtatc agagggaaca 240 gttactgatg tccatatccc ccaaaatggttcccgagccc tgttagcctt cttacaggaa 300 gccaacatcc agtacaaggt cctcatagaagatcttcaga aaacactgga gaagggaagc 360 agcttgcaca cccagagaaa ccgaagatccctctctggat ataattatga agtttatcac 420 tccttagaag aaattcaaaa ttggatgcatcatctgaata aaactcactc aggcctcatt 480 cacatgttct ctattggaag atcatatgagggaagatctc tttttatttt aaagctgggc 540 agacgatcac gactcaaaag agctgtttggatagactgtg gtattcatgc aagagaatgg 600 attggtcctg ccttttgtca gtggtttgtaaaagaagctc ttctaacata taagagtgac 660 ccagccatga gaaaaatgtt gaatcatctatatttctata tcatgcctgt gtttaacgtc 720 gatggatacc attttagttg gaccaatgatcgattttgga gaaaaacaag gtcaaggaac 780 tcaaggtttc gctgccgtgg agtggatgccaatagaaact ggaaagtgaa gtggtgtgat 840 gaaggagctt ctatgcaccc ttgtgatgacacatactgtg gcccttttcc agaatctgag 900 ccggaagtga aggctgtagc taacttccttcgaaaacaca gaaagcacat tagggcttat 960 ctctcctttc atgcatatgc tcagatgttactgtatccct attcttacaa atatgcaaca 1020 attcccaatt ttagatgtgt ggtaagtatt1050 12 350 PRT homo sapiens 12 Met Lys Cys Leu Gly Lys Arg Arg Gly GlnAla Ala Ala Phe Leu Pro 1 5 10 15 Leu Cys Trp Leu Phe Leu Lys Ile LeuGln Pro Gly His Ser His Leu 20 25 30 Tyr Asn Asn Arg Tyr Ala Gly Asp LysVal Ile Arg Phe Ile Pro Lys 35 40 45 Thr Glu Glu Glu Ala Tyr Ala Leu LysLys Ile Ser Tyr Gln Leu Lys 50 55 60 Val Asp Leu Trp Gln Pro Ser Ser IleSer Tyr Val Ser Glu Gly Thr 65 70 75 80 Val Thr Asp Val His Ile Pro GlnAsn Gly Ser Arg Ala Leu Leu Ala 85 90 95 Phe Leu Gln Glu Ala Asn Ile GlnTyr Lys Val Leu Ile Glu Asp Leu 100 105 110 Gln Lys Thr Leu Glu Lys GlySer Ser Leu His Thr Gln Arg Asn Arg 115 120 125 Arg Ser Leu Ser Gly TyrAsn Tyr Glu Val Tyr His Ser Leu Glu Glu 130 135 140 Ile Gln Asn Trp MetHis His Leu Asn Lys Thr His Ser Gly Leu Ile 145 150 155 160 His Met PheSer Ile Gly Arg Ser Tyr Glu Gly Arg Ser Leu Phe Ile 165 170 175 Leu LysLeu Gly Arg Arg Ser Arg Leu Lys Arg Ala Val Trp Ile Asp 180 185 190 CysGly Ile His Ala Arg Glu Trp Ile Gly Pro Ala Phe Cys Gln Trp 195 200 205Phe Val Lys Glu Ala Leu Leu Thr Tyr Lys Ser Asp Pro Ala Met Arg 210 215220 Lys Met Leu Asn His Leu Tyr Phe Tyr Ile Met Pro Val Phe Asn Val 225230 235 240 Asp Gly Tyr His Phe Ser Trp Thr Asn Asp Arg Phe Trp Arg LysThr 245 250 255 Arg Ser Arg Asn Ser Arg Phe Arg Cys Arg Gly Val Asp AlaAsn Arg 260 265 270 Asn Trp Lys Val Lys Trp Cys Asp Glu Gly Ala Ser MetHis Pro Cys 275 280 285 Asp Asp Thr Tyr Cys Gly Pro Phe Pro Glu Ser GluPro Glu Val Lys 290 295 300 Ala Val Ala Asn Phe Leu Arg Lys His Arg LysHis Ile Arg Ala Tyr 305 310 315 320 Leu Ser Phe His Ala Tyr Ala Gln MetLeu Leu Tyr Pro Tyr Ser Tyr 325 330 335 Lys Tyr Ala Thr Ile Pro Asn PheArg Cys Val Val Ser Ile 340 345 350 13 2128 DNA homo sapiens 13attaaagatc aggtcagctg ctgctgctgc tgctgctgct tgtcccaaga ccaagtcgta 60atagcaactt cccttcctca gctgcctgaa cttttttttt cccttgtagc tggagagaag 120tgtcacattt tgctcactct caaccttcct cgcccacccc cttcccggag aacctgtgcg 180gtgtgtagag ggtgctgtga gccacctcca gcctcgggtg gctgcttaag taactttcaa 240ctcctctctt cttaacacta tgaagtgtct cgggaagcgc aggggccagg cagctgcttt 300cctgcctctt tgctggctct ttttgaagat tctgcaaccg gggcacagcc acctttataa 360caaccgctat gctggtccac agggaaagaa acttttgaaa taataatcag ttgcctggta 420tactgctcaa tgatattgcc acacgtaaga acaagcatac agtgataaag tgataagatt 480tattcccaaa acagaagagg aagcatatgc actgaagaaa atatcctatc aacttaaggt 540tggttcctgc actacaggtg gacctgtggc agcccagcag tatctcctat gtatcagagg 600gaacagttac tgatgtccat atcccccaaa atggttcccg agccctgtta gccttcttac 660aggaagccaa catccagtac aaggtcctca tagaagatct tcagaaaaca ctggagaagg 720gaagcagctt gcacacccag agaaaccgaa gatccctctc tggatataat tatgaagttt 780atcactcctt agaagaaatt caaaattgga tgcatcatct gaataaaact cactcaggcc 840tcattcacat gttctctatt ggaagatcat atgagggaag atctcttttt attttaaagc 900tgggcagacg atcacgactc aaaagagctg tttggataga ctgtggtatt catgcaagag 960aatggattgg tcctgccttt tgtcagtggt ttgtaaaaga agtcctagaa aacacagctc 1020acaaatgtca agaatgtact aaatttacaa aatatctctg ccactaccaa aaccacaaaa 1080gtatgcttaa tcttgtaagt attgagtaat aaaattttct aaacattcct aaaaactctt 1140ctaacatata agagtgaccc agccatgaga aaaatgttga atcatctata tttctatatc 1200atgcctgtgt ttaacgtcga tggataccat tttagttgga ccaatgatcg attttggaga 1260aaaacaaggt caaggaactc aaggtttcgc tgccgtggag tggatgccaa tagaaactgg 1320aaagtgaagt ggtgtgatga aggagcttct atgcaccctt gtgatgacac atactgtggc 1380ccttttccag aatctgagcc ggaagtgaag gctgtagcta acttccttcg aaaacacaga 1440aagcacatta gggcttatct ctcctttcat gcatatgctc agatgttact gtatccctat 1500tcttacaaat atgcaacaat tcccaatttt agatgtgtgg aatctgcagc ttataaagct 1560gtgaatgcac ttcagtcagt atacggggta cgatacagat atggaccagc ctccacaacg 1620ttgtatgtga gctctggtag ctcaatggat tgggcctaca aaaatggaat accttatgca 1680tttgctttcg aactacgtga cactggatat tttggatttt tactcccaga gatgctcatc 1740aaacccacct gtacagaaac tatgctggct gtgaaaaata tcacaatgca cctgctaaag 1800aaatgtccct gagacagccc aaggctcaag tcaactgcca taggattctg agcaaggcct 1860acttggccct ggatagaaat tgttttcaaa gagaagggca gctgcttaga gtgaacatgt 1920ctatggactt taaaaagacc ccacgcaatt ttgacttttt ttggggccaa tttggaaaaa 1980acagttaagt atttgaccct gtgcatgtac atcaggcttc atgctgcttt tctgaagcta 2040agatggttct aagtactaat gataatggca aacacatgtt tgtgtttatc ctaataaata 2100ttttacatgt gaaaaaaaaa aaaaaaaa 2128

What is claimed is:
 1. An isolated nucleic acid molecule comprising at least 24 contiguous bases of nucleotide sequence first disclosed in the NHP polynucleotide described in SEQ ID NO:
 9. 2. An isolated nucleic acid molecule comprising a nucleotide sequence that: (a) encodes the amino acid sequence shown in SEQ ID NO: 10; and (b) hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 9 or the complement thereof.
 3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence disclosed in SEQ ID NO:
 12. 